EP0078331A1 - Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin - Google Patents

Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin Download PDF

Info

Publication number
EP0078331A1
EP0078331A1 EP81109312A EP81109312A EP0078331A1 EP 0078331 A1 EP0078331 A1 EP 0078331A1 EP 81109312 A EP81109312 A EP 81109312A EP 81109312 A EP81109312 A EP 81109312A EP 0078331 A1 EP0078331 A1 EP 0078331A1
Authority
EP
European Patent Office
Prior art keywords
immunoglobulin
fraction
acid
aggregate
iii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP81109312A
Other languages
English (en)
French (fr)
Other versions
EP0078331B1 (de
Inventor
Yahiro Uemura
Takashi Goto
Yoshiaki Kano
Satoshi Funakoshi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsubishi Tanabe Pharma Corp
Original Assignee
Green Cross Corp Japan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Green Cross Corp Japan filed Critical Green Cross Corp Japan
Priority to EP81109312A priority Critical patent/EP0078331B1/de
Priority to AT81109312T priority patent/ATE17654T1/de
Priority to DE8181109312T priority patent/DE3173666D1/de
Publication of EP0078331A1 publication Critical patent/EP0078331A1/de
Application granted granted Critical
Publication of EP0078331B1 publication Critical patent/EP0078331B1/de
Priority to HK955/86A priority patent/HK95586A/xx
Expired legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065Purification, fragmentation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • This invention relates to a process for preparing immunoglobulin suitable for intravenous injection.
  • the immunoglobulin Since the immunoglobulin possesses antibody activity to many pathogens, it is administered to patients deficient in various antibodies to protect them from injection diseases or for therepeutic purposes.
  • the immunoglobulin preparations have been developed in two types, one being for intravenous injection and the other for intramuscular injection, and both are widely in clinical use.
  • the known methods for the preparation of the intravenous administration immunoglobulin type/in include that in which crude immunoglobulin is treated with proteases such as pepsin and plasmin, that which employs chemical modification of immunoglobulin by acylation and other chemical means, and that utilizing fractionation of the plasma or of Cohn's plasma fractions using polyethylene glycol, Pluronics (polyoxyethylene-polyoxypropylene copolymer, Wyandotte Chemicals Co.), or the like.
  • the fractionation with polyethylene glycol or Pluronics is generally believed to be the most desirable means to recover the immunoglobulin in the form existing in the living body, that is, unmodified and undecomposed immunoglobulin.
  • the procedure has already been disclosed in detail by Polson and Coval [Japanese Patent Application "Kokai” (Laid-open) Nos. 46,814/1975, 91,321/1976 and 20,415/1978].
  • the polyethylene glycol or Pluronic fractionation is carried out under known conditions, it is possible to obtain an immunoglobulin preparation containing no aggregate-type immunoglobulin and suitable for intravenous injection, but the yield is always low. Therefore, an improvement in the yield has been eagerly awaited.
  • the present inventors conducted sttdies to elucidate the cause of- the decrease in yield and as a result found that in the step of removing the aggregate-type immunoglobulin at a low concentration of polyethylene glycol or a Pluronic® added to the raw immunoglobulin material, the non-aggregate-type immunoglobulin is also - removed at the same time. Further, it. was also found that an immunoglobulin material containing a comparatively large amount of aggregate-type immunoglobulin is fractio- nated with polyethylene glycol or a Pluronic®, the loss in non-aggregate-type immunoglobulin also becomes great.
  • the present inventors further made an effort to solve the novel technical problem associated with the aforesaid difficulty inherent in the prior art and, as a result, found that the yield of non-aggregate-type immunoglobulin is remarkably improved by treating the raw immunoglobulin material with an acid to dissociate the aggregate-type immunoglobulin and then subjecting the material to the fractionation with polyethylene glycol or a polyoxyethylene-polyoxypropylene copolymer. Based on this finding, the present invention has been accomplished.
  • An object of this invention is to provide a process for preparing in a high yield an immunoglobulin suitable for use in intravenous injection from a plasma or Cohn's fraction I+II+III, fraction II+III, fraction II, or fraction III obtained by subjecting a plasma to the Cohn's cold alcohol fractionation.
  • a process for preparing an immunoglobulin suitable for intravenous injection which comprises treating a plasma or the Cohn's fraction I+II+III, fraction II+III, fraction II, or fraction III obtained by subjecting a plasma to Cohn's cold alcohol fractionation with an acid at pH 3.2 to 5.0 and at 4° to 15°C for 30 to 180 minutes, adding to the resulting material at pH 4.6 to 5.4 an alkylene oxide polymer or copolymer having a molecular weight of 2,000 to 20,000 to a concentration of 4.5 to 5.5% (W/V), removing an aggregate-type immunoglobulin as a precipitate, and adding again said polymer or copolymer at pH 8.0 to 9.0 to a.concentration of 6 to 13% (W/V) to recover as a precipitate a non-aggregate-type immunoglobulin containing substantially no aggregate-type immunoglobulin.
  • the plasma used as starting material according to this invention is preferably that originated from human blood in view of the problem of antigenicity.
  • the Cohn's plasma fractions I+II+III, II+III, II, and III are substantially a-, ⁇ - and y-globulin (IgG, IgA, IgM).
  • the plasma y-globulin fraction is obtained by the continuous precipitation with cold ethanol as described in detail in Journal of Clinical Investigation, 23, 417 (19.44) and Journal of the American Chemical Society, 68, 479 (1946).
  • the acid treatment is carried out by keeping the starting material under acidic conditions.
  • the aggregate-type immunoglobulin in the raw material is dissociated first into a non-aggregate type.
  • the acid conditions are pH 3,2 to 5.0, preferably 3.8 to 4.2 and an ionic strength of 0.002 to 0.30, preferably 0.10 to 0.20.
  • Other pH conditions are undesirable because if pH is below 3.2, denaturation of the protein will take place, while if it is above 5.0, the said dissociation of the aggregate-type immonoglobulin becomes insufficient.
  • the concentration of protein is not critical, but is preferably 2 to 10% (W/V), because of the ease of operation.
  • the temperature of acid treatment is 4° to 15°C.
  • the duration of the acid treatment is 30 to 180 minutes. If the duration of treatment is shorter than 30 minutes, the aforesaid dissociation becomes insufficient, while too long a duration of treatment is a waste of time and sometimes even gives unfavorable results.
  • the acids used for the treatment include inorganic acids such as hydrochloric acid and phosphoric acid and organic acids such as acetic acid and citric acid.
  • a fractionation is performed using an alkylene oxide polymer or copolymer having a molecular weight of 2,000 to 20,000, to produce a high-purity non-aggregate-type immunoglobulin in a high yield.
  • the alkylene group of the alkylene oxide polymer used in the fractionation is one having 1 to 4 carbon atoms such as methylene, ethylene, propylene or butylene group.
  • the alkylene oxide copolymers include copolymers of two or more alkylene oxides such as polyoxyethylene-polyoxypropylene copolymer.
  • the present inventors found that in the fractionation using an alkylene oxide polymer or copolymer having a molecular weight of 2,000 to 20,000, the pH conditions area very important factor and the yield and purity of the immunoglobulin are markedly improved only in a very limited pH range of from 4.6 to 5.4, preferably from 4.8 to 5.2.
  • the above polymer or copolymer is added to the starting material to a concentration of 4.5 to 5.5% , preferably 5% (W/V) and the precipitated impurities, e.g. aggregate-type immunoglobulin, are removed by . customary means, e.g. centrifuging (1,000-5,000 rpm). To the supernatant thus fractionated is further added the above polymer or copolymer to a concentration of 6 to 13%. By adjusting pH to 8.0 to 9.0, the intended non-aggregate-type immunoglobulin is precipitated and can be recovered by customary means, e.g. centrifuging (1,000 to 5,000 rpm). The recovery of the intended product under the above conditions is 60% or more.
  • customary means e.g. centrifuging (1,000 to 5,000 rpm).
  • the precipitate which was formed is again dissolved, for example, in a physiological saline or a 0.02 M acetate buffer solution admixed with 0.6% of sodium chloride, 2% of mannit and 1% of albumin, and the resulting solution is passed through a bacterial filter to obtain an intravenous immunoglobulin solution suitable for clinical use.
  • the immunoglobulin in this solution shows no change in its properties upon dispensing the solution in small portions into vials and lyophilizing. Accordingly, when the product is intended for long-term storage, it can be made into the form of lyophilized preparation.
  • the immunoglobulin prepared by the present process contains substantially no aggregate-type immunoglobulin and the anticomplementary activity is less than 20 units, as assayed on a solution of 5% in concentration, the purity as IgG being 90% or above.
  • the recovery of immunoglobulin was determined by single immunodiffusion and the purity by electrophoresis using a cellulose acetate membrane.
  • the anticomplementary activity was assayed by the method of Kobat and Mayer [Experimental Immunochemistry, p 225 (1961)] and the method of Nishioka and Okada ["Biochemistry of Immunity", p 103 (1971), Kyoritsu Publishing Company].
  • the Cohn's fraction II+III paste.(1 kg) obtained by the cold alcohol fractionation was dissolved in 10 liters of a 0.6% aqueous sodium chloride solution.
  • the solution was adjusted to pH 3.8 with IN hydrochloric acid and stirred at 4°c for 60 minutes to effect acid treatment.
  • To the solution was added 500 g of polyethylene glycol (average molecular weight 4,000). While allowing the polyethylene glycol to dissolve, pH of the solution was gradually increased with 1N aqueous sodium hydroxide solution. As soon as the pH reached 5.0, the precipitate was removed by centrifugation (4,000 rpm) to obtain a clear supernatant.
  • the recovery of IgG in the supernatant was 85% based on the fraction II+III and the purity of IgG was 97%.
  • the immunoglobulin precipitated under the above conditions was recovered by centrifugation (4,000 rpm). The whole of the recovered precipitate was dissolved in a 0.02 M acetate buffer solution (pH 6.6) containing 1% of human albumin and using the same solvent the concentration was adjusted to 5%.
  • the solution was sterilized by passing through a Millipore® filter (Millipore Co.) and aseptically dispensed in small containers. One half of the dispensed liquor was immediately lyophilized to yield. a dry preparation.
  • the ultimate yield of IgG from the starting material was 84%, as contrasted with 63% when the acid treatment at pH 3.8 to the starting material was omitted.
  • the purity was 95%, excluding the albumin which was added afterwards.
  • the anticomplementary activities (at a protein concentration of 5%) of the liquid preparation and the solution of lyophilized preparation were found to be 14 and 16, respectively.
  • a 5% solution was administered to 5 mice, about 20 g in body weight, at a dose of 1 ml per mouse. Neither decrease in body weight nor any anomaly in Pilo- erection was noted during an observation period of one week.
  • the lyophilized preparation was tested after one year of storage at 4°C, but no change in solubility and anticomplementary activity was observed as compared with the initial preparation.
  • a Cohn's fraction II paste (500 g) obtained by the cold ethanol fractionation was dissolved in 10 liters of a 0.1% sodium chloride solution. Immunoglobulin containing no aggregate type was recovered from the solution in a manner similar to that in Example 1. The yield in terms of IgG was 80%, as contrasted with'62% when the acid treatment (pH 3.8) was omitted. The purity was 97%.
  • the immunoglobulin obtained above was dissolved in a 0.5% sodium chloride solution to a protein concentration of 5%. After addition of 1% of mannit, the solution was passed through a bacterial filter and lyophilized. The lyophilized preparation was dissolved in distilled water for injection to a protein concentration of 5%. The anticomplementary activity at this concentration was found to be 13.
  • Example 1 The procedure of Example 1 was repeated, except that the polyethylene glycol (average molecular weight 4,000) was replaced by the same amount of a polyoxyethylene-polyoxypropylene copolymer (average molecular weight 15,000). The immunoglobulin was recovered with the same results as in Example 1.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
EP81109312A 1981-10-29 1981-10-29 Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin Expired EP0078331B1 (de)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP81109312A EP0078331B1 (de) 1981-10-29 1981-10-29 Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin
AT81109312T ATE17654T1 (de) 1981-10-29 1981-10-29 Verfahren zur herstellung von intravenoesem injizierbarem immunoglobulin.
DE8181109312T DE3173666D1 (en) 1981-10-29 1981-10-29 Process for preparing immunoglobulin suitable for intravenous injection
HK955/86A HK95586A (en) 1981-10-29 1986-12-11 Process for preparing immunoglobulin suitable for intravenous injection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP81109312A EP0078331B1 (de) 1981-10-29 1981-10-29 Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin

Publications (2)

Publication Number Publication Date
EP0078331A1 true EP0078331A1 (de) 1983-05-11
EP0078331B1 EP0078331B1 (de) 1986-01-29

Family

ID=8187984

Family Applications (1)

Application Number Title Priority Date Filing Date
EP81109312A Expired EP0078331B1 (de) 1981-10-29 1981-10-29 Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin

Country Status (4)

Country Link
EP (1) EP0078331B1 (de)
AT (1) ATE17654T1 (de)
DE (1) DE3173666D1 (de)
HK (1) HK95586A (de)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0092186A2 (de) * 1982-04-16 1983-10-26 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Verfahren zur Herstellung von intravenös zu verabreichendem Gammaglobulin
EP0168506A1 (de) * 1984-07-07 1986-01-22 Armour Pharma GmbH Verfahren zur Herstellung von gamma-Globulin zur intravenösen Anwendung
US4835257A (en) * 1984-07-07 1989-05-30 Armour Pharma Gmbh Process for preparing gamma globulin suitable for intravenous administration using peg and a citrate buffer
WO2016012803A1 (en) * 2014-07-25 2016-01-28 Bio Products Laboratory Limited Improved process for the preparation of immunoglobulin g (igg)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3597409A (en) * 1970-05-25 1971-08-03 American Cyanamid Co Process for recoverring immunoglobulin a and immunoglobulin m
US3763135A (en) * 1971-11-08 1973-10-02 Baxter Laboratories Inc Gamma globulin production from cohn fraction iii using polyethylene glycol
US3808189A (en) * 1973-03-15 1974-04-30 American Cyanamid Co Isolation of gamma globulin preparations enriched in iga and igm using polyethylene glycol
DE2364792A1 (de) * 1973-01-15 1974-07-18 South African Inventions Verfahren zum reinigen von gammaglobulin
GB1435816A (en) * 1972-11-27 1976-05-19 Baxter Laboratories Inc Production of gamma globulin
US4000121A (en) * 1973-01-30 1976-12-28 Baxter Travenol Laboratories, Inc. Production of antisera comprising fractionating plasma or serum with an ethylene oxide-polyoxypropylene block copolymer
US4124576A (en) * 1976-12-03 1978-11-07 Coval M L Method of producing intravenously injectable gamma globulin
US4164495A (en) * 1976-04-06 1979-08-14 Nordisk Insulinlaboratorium Method of recovering immunoglobulin using a polyol and an alkanoic acid
US4165370A (en) * 1976-05-21 1979-08-21 Coval M L Injectable gamma globulin

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3597409A (en) * 1970-05-25 1971-08-03 American Cyanamid Co Process for recoverring immunoglobulin a and immunoglobulin m
US3763135A (en) * 1971-11-08 1973-10-02 Baxter Laboratories Inc Gamma globulin production from cohn fraction iii using polyethylene glycol
GB1435816A (en) * 1972-11-27 1976-05-19 Baxter Laboratories Inc Production of gamma globulin
DE2364792A1 (de) * 1973-01-15 1974-07-18 South African Inventions Verfahren zum reinigen von gammaglobulin
US4000121A (en) * 1973-01-30 1976-12-28 Baxter Travenol Laboratories, Inc. Production of antisera comprising fractionating plasma or serum with an ethylene oxide-polyoxypropylene block copolymer
US3808189A (en) * 1973-03-15 1974-04-30 American Cyanamid Co Isolation of gamma globulin preparations enriched in iga and igm using polyethylene glycol
US4164495A (en) * 1976-04-06 1979-08-14 Nordisk Insulinlaboratorium Method of recovering immunoglobulin using a polyol and an alkanoic acid
US4165370A (en) * 1976-05-21 1979-08-21 Coval M L Injectable gamma globulin
US4124576A (en) * 1976-12-03 1978-11-07 Coval M L Method of producing intravenously injectable gamma globulin

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0092186A2 (de) * 1982-04-16 1983-10-26 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Verfahren zur Herstellung von intravenös zu verabreichendem Gammaglobulin
EP0092186A3 (en) * 1982-04-16 1984-07-18 Fujirebio Kabushiki Kaisha Also Trading As Fujirebio Inc. Method of preparing gamma globulin suitable for intravenous administration
EP0168506A1 (de) * 1984-07-07 1986-01-22 Armour Pharma GmbH Verfahren zur Herstellung von gamma-Globulin zur intravenösen Anwendung
WO1986000530A1 (en) * 1984-07-07 1986-01-30 Woelm Pharma Gmbh & Co. Process for preparing gamma globulin suitable for intravenous administration
US4835257A (en) * 1984-07-07 1989-05-30 Armour Pharma Gmbh Process for preparing gamma globulin suitable for intravenous administration using peg and a citrate buffer
WO2016012803A1 (en) * 2014-07-25 2016-01-28 Bio Products Laboratory Limited Improved process for the preparation of immunoglobulin g (igg)
US11149079B2 (en) 2014-07-25 2021-10-19 Bio Products Laboratory Limited Process for the preparation of immunoglobulin G (IgG)

Also Published As

Publication number Publication date
DE3173666D1 (en) 1986-03-13
EP0078331B1 (de) 1986-01-29
ATE17654T1 (de) 1986-02-15
HK95586A (en) 1986-12-19

Similar Documents

Publication Publication Date Title
US4371520A (en) Process for preparing immunoglobulin suitable for intravenous injection
EP0073371B1 (de) Intravenös injektierbares Globulin aus Immunserum und Verfahren zu seiner Herstellung
CA1245155A (en) Immunoglobulin-g-containing fraction
US4276283A (en) Method of preparing an intravenously administrable immune globulin preparation containing antibodies and preparations produced according to this method
US4216205A (en) Process of preparing a serum protein composition for intravenous application
KR20000015935A (ko) 농축 항체 제제
IE46104B1 (en) Fractionation process
US4168303A (en) Lyophilized native gamma globulin preparation for intravenous administration
CA1137413A (en) Method for the production of immunoglobulin having a high monomer content
US3916026A (en) Method for the preparation of gamma-globulin suitable for intravenous use
US4164495A (en) Method of recovering immunoglobulin using a polyol and an alkanoic acid
US5075425A (en) Process for the preparation of a pharmaceutical which contains igg, iga and igm and can be administered intravenously
US5648472A (en) Process for preparing virus-inactivated immunoglobulin solutions
CA2232420A1 (en) Immunoglobulin preparation and preparation process thereof
JPS62174027A (ja) 改良されたキモパパイン及び製薬学的組成物
EP0078331B1 (de) Verfahren zur Herstellung von intravenösem injizierbarem Immunoglobulin
USRE31268E (en) Method of recovering immunoglobulin using a polyol and an alkanoic acid
JPH0365327B2 (de)
EP0123375A1 (de) Herstellung von gamma-Globulin zur intravenösen Darreichung
EP0339919B1 (de) Methode zur Reinigung von Antithrombin-III und Antithrombin-III-Zusammensetzung enthaltend dieses Antithrombin-III
CA1172166A (en) Process for preparing immunoglobulin suitable for intravenous injection
JPS6221359B2 (de)
KR830002739B1 (ko) 정맥주사용 감마글로블린의 제조방법
AU2016231646B2 (en) Method for the preparation of immunoglobulins
JP2678249B2 (ja) アンチトロンビン−▲iii▼製剤

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB LI LU NL SE

RBV Designated contracting states (corrected)

Designated state(s): AT BE CH DE FR GB LI LU NL SE

17P Request for examination filed

Effective date: 19831011

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Designated state(s): AT BE CH DE FR GB LI LU NL SE

REF Corresponds to:

Ref document number: 17654

Country of ref document: AT

Date of ref document: 19860215

Kind code of ref document: T

REF Corresponds to:

Ref document number: 3173666

Country of ref document: DE

Date of ref document: 19860313

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed
EPTA Lu: last paid annual fee
PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LU

Payment date: 19941001

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 19941011

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 19941012

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 19941017

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 19941019

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 19941021

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 19941025

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 19941031

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 19941208

Year of fee payment: 14

EAL Se: european patent in force in sweden

Ref document number: 81109312.9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19951029

Ref country code: GB

Effective date: 19951029

Ref country code: AT

Effective date: 19951029

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Effective date: 19951030

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Effective date: 19951031

Ref country code: CH

Effective date: 19951031

Ref country code: BE

Effective date: 19951031

BERE Be: lapsed

Owner name: THE GREEN CROSS CORP.

Effective date: 19951031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19960501

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 19951029

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Effective date: 19960628

EUG Se: european patent has lapsed

Ref document number: 81109312.9

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee

Effective date: 19960501

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19960801

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST